Isolated nucleic acid molecules encoding cancer associated antigens, the antigens per se, and uses thereof

ABSTRACT

The invention relates to newly identified cancer associated antigens. It has been discovered that each of these molecules provokes antibodies when expressed by a subject. The ranifications of this observation are also a part of this invention.

RELATED APPLICATIONS

[0001] This application is a continuation in part of Ser. No. 09/602,362, filed Jun. 22, 2000 which is a continuation in part of Ser. No.09/451,739, filed Nov. 30, 1999, both of which are incorporated byreference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to antigens associated with cancer, thenucleic acid molecules encoding them, as well as the uses of these.

BACKGROUND AND PRIOR ART

[0003] It is fairly well established that many pathological conditions,such as infections, cancer, autoimmune disorders, etc., arecharacterized by the inappropriate expression of certain molecules.These molecules thus serve as “markers” for a particular pathological orabnormal condition. Apart from their use as diagnostic “targets”, i.e.,materials to be identified to diagnose these abnormal conditions, themolecules serve as reagents which can be used to generate diagnosticand/or therapeutic agents. A by no means limiting example of this is theuse of cancer markers to produce antibodies specific to a particularmarker. Yet another non-limiting example is the use of a peptide whichcomplexes with an MHC molecule, to generate cytolytic T cells againstabnormal cells.

[0004] Preparation of such materials, of course, presupposes a source ofthe reagents used to generate these. Purification from cells is onelaborious, far from sure method of doing so. Another preferred method isthe isolation of nucleic acid molecules which encode a particularmarker, followed by the use of the isolated encoding molecule to expressthe desired molecule.

[0005] Two basic strategies have been employed for the detection of suchantigens, in e.g., human tumors. These will be referred to as thegenetic approach and the biochemical approach. The genetic approach isexemplified by, e.g., dePlaen et al., Proc. Natl. Sci. USA 85: 2275(1988), incorporated by reference. In this approach, several hundredpools of plasmids of a cDNA library obtained from a tumor aretransfected into recipient cells, such as COS cells, or intoantigen-negative variants of tumor cell lines which are tested for theexpression of the specific antigen. The biochemical approach,exemplified by, e.g., O. Mandelboim, et al., Nature 369: 69 (1994)incorporated by reference, is based on acidic elution of peptides whichhave bound to MHC-class I molecules of tumor cells, followed byreversed-phase high performance liquid chromography (HPLC). Antigenicpeptides are identified after they bind to empty MHC-class I moleculesof mutant cell lines, defective in antigen processing, and inducespecific reactions with cytotoxic T-lymphocytes. These reactions includeinduction of CTL proliferation, TNF release, and lysis of target cells,measurable in an MTT assay, or a ⁵¹Cr release assay.

[0006] These two approaches to the molecular definition of antigens havethe following disadvantages: first, they are enormously cumbersome,time-consuming and expensive; and second, they depend on theestablishment of cytotoxic T cell lines (CTLs) with predefinedspecificity.

[0007] The problems inherent to the two known approaches for theidentification and molecular definition of antigens is best demonstratedby the fact that both methods have, so far, succeeded in defining onlyvery few new antigens in human tumors. See, e.g., van der Bruggen etal., Science 254: 1643-1647 (1991); Brichard et al., J. Exp. Med. 178:489-495 (1993); Coulie, et al., J. Exp. Med. 180: 35-42(1994); Kawakami,et al., Proc. Natl. Acad. Sci. USA 91: 3515-3519 (1994).

[0008] Further, the methodologies described rely on the availability ofestablished, permanent cell lines of the cancer type underconsideration. It is very difficult to establish cell lines from certaincancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg.Clin. North. Am. 10: 607-637 (1990). It is also known that someepithelial cell type cancers are poorly susceptible to CTLs in vitro,precluding routine analysis. These problems have stimulated the art todevelop additional methodologies for identifying cancer associatedantigens.

[0009] One key methodology is described by Sahin, et al., Proc. Natl.Acad. Sci. USA 92: 11810-11913 (1995), incorporated by reference. Also,see U.S. Pat. No. 5,698,396, and application Ser. No. 08/479,328, filedon Jun. 7, 1995 and Jan. 3, 1996, respectively. All three of thesereferences are incorporated by reference. To summarize, the methodinvolves the expression of cDNA libraries in a prokaryotic host. (Thelibraries are secured from a tumor sample). The expressed libraries arethen immunoscreened with absorbed and diluted sera, in order to detectthose antigens which elicit high titer humoral responses. Thismethodology is known as the SEREX method (“Serological identification ofantigens by Recombinant Expression Cloning”). The methodology has beenemployed to confirm expression of previously identified tumor associatedantigens, as well as to detect new ones. See the above referenced patentapplications and Sahin, et al., supra as well as Crew, et al., EMBO J144: 2333-2340 (1995).

[0010] This methodology has been applied to a range of tumor types,including those described by Sahin et al., supra, and Pfreundschuh,supra, as well as to esophageal cancer (Chen et al., Proc. Natl. Acad.Sci. USA 94: 1914-1918 (1997)); lung cancer (Güre. et al., Cancer Res.58: 1034-1041 (1998)); colon cancer (Ser. No. 08/948, 705 filed Oct. 10,1997) incorporated by reference, and so forth. Among the antigensidentified via SEREX are the SSX2 molecule (Sahin et al., Proc. Natl.Acad. Sci. USA 92: 11810-11813 (1995); Tureci et al., Cancer Res. 56:4766-4772 (1996); NY-ESO-1 Chen, et al., Proc. Natl. Acad. Sci. USA 94:1914-1918 (1997); and SCP1 (Ser. No. 08/892,705 filed Jul. 15, 1997)incorporated by reference. Analysis of SEREX identified antigens hasshown overlap between SEREX defined and CTL defined antigens. MAGE-1,tyrosinase, and NY-ESO-1 have all been shown to be recognized by patientantibodies as well as CTLs, showing that humoral and cell mediatedresponses do act in concert.

[0011] It is clear from this summary that identification of relevantantigens via SEREX is a desirable aim. The inventors have applied thismethodology and have identified several new antigens associated withcancer, as detailed in the description which follows.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

[0012] The SEREX methodology, as described by, e.g. Sahin, et al., Proc.Natl. Acad. Sci. USA 92: 11810-11813 (1995); Chen, et al., Proc. Natl.Acad. Sci. USA 94: 1914-1918 (1997), and U.S. Pat. No. 5,698,396, all ofwhich are incorporated by reference. In brief, total RNA was extractedfrom a sample of a cutaneous metastasis of a breast cancer patient(referred to as “BR11” hereafter), using standard CsCl guanidinethiocyanate gradient methodologies. A cDNA library was then prepared,using commercially available kits designed for this purpose. Followingthe SEREX methodology referred to supra, this cDNA expression librarywas amplified, and screened with either autologous BR11 serum which hadbeen diluted to 1:200, or with allogeneic, pooled serum, obtained from 7different breast cancer patients, which had been diluted to 1:1000. Tocarry out the screen, serum samples were first diluted to 1:10, and thenpreabsorbed with lysates of E. coli that had been transfected with nakedvector, and the serum samples were then diluted to the levels describedsupra. The final dilutions were incubated overnight at room temperaturewith nitrocellulose membranes containing phage plaques, at a density of4-5000 plaque forming units (“pfus”) per 130 mm plate.

[0013] Nitrocellulose filters were washed, and incubated with alkalinephosphatase conjugated, goat anti-human Fcγ secondary antibodies, andreactive phage plaques were visualized via incubation with5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium.

[0014] This procedure was also carried out on a normal testicular cDNAlibrary, using a 1:200 serum dilution.

[0015] A total of 1.12×10⁶ pfus were screened in the breast cancer cDNAlibrary, and 38 positive clones were identified. With respect to thetesticular library, 4×10⁵ pfus were screened, and 28 positive cloneswere identified.

[0016] Additionally, 8×10⁵ pfus from the BR11 cDNA library were screenedusing the pooled serum described. Of these, 23 were positive.

[0017] The positive clones were subcloned, purified, and excised toforms suitable for insertion in plasmids. Following amplification of theplasmids, DNA inserts were evaluated via restriction mapping(EcoRI-XbaI), and clones which represented different cDNA inserts weresequenced using standard methodologies.

[0018] If sequences were identical to sequences found in GenBank, theywere classified as known genes, while sequences which shared identityonly with ESTs, or were identical to nothing in these data bases, weredesignated as unknown genes. Of the clones from the breast cancerlibrary which were positive with autologous serum, 3 were unknown genes.Of the remaining 35, 15 were identical to either NY-ESO-1, or SSX2, twoknown members of the CT antigen family described supra while theremaining clones corresponded to 14 known genes. Of the testicularlibrary, 12 of the clones were SSX2.

[0019] The NY-ESO-1 antigen was not found, probably because thecommercial library that was used had been size fractionated to have anaverage length of 1.5 kilobases, which is larger than full lengthNY-ESO-1 cDNA which is about 750 base pairs long.

[0020] With respect to the screening carried out with pooled, allogeneicsera, four of the clones were NY-ESO-1. No other CT antigens wereidentified. With the exception of NY-ESO-1, all of the genes identifiedwere expressed universally in normal tissue.

[0021] A full listing of the isolated genes, and their frequency ofoccurrence follows, in tables 1, 2 and 3. Two genes were found in boththe BR11 and testicular libraries, i.e., poly (ADP-ribose) polymerase,and tumor suppression gene ING1. The poly (ADP-ribose) polymerase genehas also been found in colon cancer libraries screened via SEREX, as isdisclosed by Scanlan, et al., Int. J. Cancer 76: 652-58 (1998) when thegenes identified in the screening of the BR11 cDNA library by autologousand allogeneic sera were compared, NY-ESO-1 and human keratin. TABLE 1SEREX-defined genes identified by autologous screening of BR11 cDNAlibrary Gene group No. of clones Comments Expression CT genes 10NY-ESO-1 tumor, testis 5 SSX2 tumor, testis Non-CT genes 5 NuclearReceptor Co-Repressor ubiquitous 4 Poly(ADP-ribose) polymeraseubiquitous 2 Adenylosuccinatelyase ubiquitous 2 cosmid 313 (human) ESTs:muscle, brain, breast 1 CD 151 (transmembrane protein) ubiquitous 1Human HRY Gen RT-PCR: multiple normal tissues 1 Alanyl-t-RNA-Synthetaseubiquitous 1 NAD(+) ADP-Ribosyltransferase ubiquitous 1 Human keratin 10ESTs: multiple normal tissues 1 Human EGFR kinase substrate ubiquitous 1ING 1 Tumor suppressor gene RT-PCR multiple normal tissues 1 Unknowngene, ESTs: pancreas, liver, spleen, uterus NCI_CGAP_Pr12 cDNA clone 1Unknown gene ESTs: multiple normal tissues 1 Unknown gene RT-PCR:multiple normal tissues

[0022] TABLE 2 SEREX-defined genes identified by allogeneic screening ofBR11 cDNA library Gene group No. of clones Comments Expression CT genes4 NY-ESO-1 tumor, testis Non-CT genes 6 zinc-finger helicase ESTs:brain, fetal heart, total fetus 4 Acetoacetyl-CoA-thiolase ubiquitous 3KIAA0330 gene ESTs: multiple normal tissues 2 U1snRNP ubiquitous 1 Humanaldolase A ubiquitous 1 Retinoblastoma binding protein 6 ESTs: tonsils,fetal brain, endothelial cells, brain 1 α2-Macroglobulin receptorubiquitous associatad protein 1 Human Keratin 10 ESTs: multiple normaltissues

[0023] TABLE 3 SEREX-defined genes identified by screening of atesticular cDNA library with BR11 serum Gene group No. of clonesComments Expression CT genes: 12 SSX2 tumor, testis Non-CT genes: 3Rho-associated coiled-coil ubiquitous forming protein 3 Poly(ADP-ribose)polymerase ubiquitous 3 Gene from HeLa cell, similar to ubiquitous TITIN2 Gene from parathyroid tumor RT-PCR: multiple normal tissues 1Transcription termination factor ubiquitous I-interacting peptide 21 1Gene from fetal heart ESTs: multiple normal tissues 1 ING 1 tumorsuppressor gene RT-PCR multiple normal tissues 1 KIAA0647 cDNA ESTs:multiple normal tissues 1 KIAA0667 cDNA ESTs: multiple normal tissues

EXAMPLE 2

[0024] The mRNA expression pattern of the cDNAs identified in example 1,in both normal and malignant tissues, was studied. To do this, genespecific oligonucleotide primers were designed which would amplify cDNAsegments 300-600 base pairs in length, using a primer meltingtemperature. The primers used for amplifying MAGE-1,2,3 and 4, BAGE,NY-ESO-1, SCP1, 2, 3, 4 and 5 were known primers, or were based onpublished sequences. See Chen, et al. supra; Tureci, et al., Proc. Natl.Acad. Sci. USA 95: 5211-16 (1998). Gure, et al., Int. J. Cancer 72:965-71(1997); Chen, et al., Proc. Natl. Acad. Sci. USA 91: 1004-1008(1994); Gaugler, et al., J. Exp. Med. 179: 921-930 (1994), dePlaen, etal., Immunogenetics 40: 360-369 (1994), of which are incorporated byreference. RT-PCR was carried out for 35 amplification cycles, at anannealing temperature of 60° C. Using this RT-PCR assay, the breastcancer tumor specimen was positive for a broad range of CT antigens,including MAGE-1,3 AND 4, BAGE, SSX2, NY-ESO-1 and CT7. The known CTantigens SCP-1, SSX1, 4 and 5 were not found to be expressed.

[0025] An additional set of experiments were carried out, in which theseroreactivity of patient sera against tumor antigens was tested.Specially, ELISAs were carried out, in accordance with Stockert, et al.,J. Exp. Med. 187: 1349-11354 (1998), incorporated by reference, todetermine if antibodies were present in the patient sera. Assays wererun for MAGE-1, MAGE-3, NY-ESO-1, and SSX2. The ELISAs were positive forNY-ESO-1 and SSX2, but not the two MAGE antigens.

EXAMPLE 3

[0026] Two clones (one from the breast cancer cDNA library and one fromthe testicular library), were identified as a gene referred to as ING1,which is a tumor suppressor gene candidate. See Garkavtsev, et al.,Nature 391: 295-8 (1998), incorporated by reference. The sequence foundin the breast cancer library, differed from the known sequence of ING1at six residues, i.e., positions 818, 836, 855, 861, 866 and 874. Thesequence with the six variants is set forth at SEQ ID NO: 1. Thesequence of wild type ING1 is set out at SEQ ID NO: 2.

[0027] To determine if any of these differences represented a mutationin tumors, a short, PCR fragment which contained the six positionsreferred to supra was amplified from a panel of allogeneic normaltissue, subcloned, amplified, and sequenced following standard methods.

[0028] The results indicated that the sequences in the allogeneictissues were identical to what was found in tumors, ruling out thehypothesis that the sequence differences were a tumor associatedmutation. This conclusion was confirmed, using the testicular libraryclone, and using restriction analysis of ING1 cDNA taken from normaltissues. One must conclude, therefore, that the sequence informationprovided by Garkavtsev, et al., supra, is correct.

EXAMPLE 4

[0029] Additional experiments were carried out to determine whethergenetic variations might exist in the 5′ portion of the ING1 gene, whichmight differ from the 5′ portion of the clone discussed supra (SEQ IDNO: 1). In a first group of experiments, attempts were made to obtainfull length ING1 cDNA from both the breast tumor library, and thetesticular library. SEQ ID NO: 1 was used as a probe of the library,using standard methods.

[0030] Four clones were isolated from the testicular library and nonewere isolated from the breast cancer library. The four clones, followingsequencing, were found to derive from three transcript variants. Thethree variants were identical from position 586 down to their 3′ end,but differed in their 5′ regions, suggesting alternatively splicedvariants, involving the same exon-intron junction. All three differedfrom the sequence of ING1 described by Garkavtsev, et al., in Nat.Genet. 14: 415-420 (1996). These three variants are set out as SEQ IDNOS: 1, 3 and 4.

[0031] All of the sequences were then analyzed. The ORFs of SEQ ID NOS:2, 1 and 4 (SEQ ID NO: 2 is the originally disclosed, ING1 sequence),encode polypeptides of 294, 279 and 235 amino acids, of which 233 areencoded by the 3′ region common to the three sequences. These putativesequences are set out as SEQ ID NOS:19, 5, and 7. With respect to SEQ IDNO: 3, however, no translational initiation site could be identified inits 5′ region.

EXAMPLE 5

[0032] The data regarding SEQ ID NO: 3, described supra, suggestedfurther experiments to find additional ORFs in the 5-end of varianttranscripts of the molecule. In order to determine this, 5 ′-RACE -PCRwas carried out using gene specific and adapted specific primers,together with commercially available products, and standardmethodologies.

[0033] The primers used for these experiments were: (SEQ ID NOS: 9 and10), for SEQ ID NO: 1 CACACAGGATCCATGTTGAGTCCTGCCAACGGCGTGGTCGTGGTTGCTGGACGCG; (SEQ ID NOS: 11 and 12), for SEQ ID NO: 3CCCAGCGGCCCTGACGCTGTC CGTGGTCGTGGTTGCTGGACGCG; and (SEQ ID NOS: 13 and14), for SEQ ID NO: 4 GGAAGAGATAAGGCCTAGGGAAG CGTGGTCGTGGTTGCTGGACGCG.

[0034] Cloning and sequencing of the products of RACE PCR showed thatthe variant sequence of SEQ ID NO: 4 was 5′ to SEQ ID NO: 3, and thatfull length cDNA for the variant SEQ ID NO: 3 contained an additionalexon 609 nucleotides long, positioned between SEQ ID NO: 3 and theshared, 3′ sequence referred to supra. This exon did not include an ORF.The first available initiation site would be an initial methionine atamino acid 70 of SEQ ID NO: 1. Thus, if expressed, SEQ ID NO: 3 wouldcorrespond to a molecule with a 681 base pair, untranslated 5′ end and aregion encoding 210 amino acids (SEQ ID NO:6).

EXAMPLE 6

[0035] The presence of transcript variants with at least 3 differenttrancriptional initiation sites, and possibly different promoters,suggested that mRNA expression might be under different, tissue specificregulation.

[0036] To determine this, variant-specific primers were synthesized, andRT-PCR was carried out on a panel of tissues, using standard methods.

[0037] SEQ ID NO: 1 was found to be expressed universally in all of thenormal breast, brain and testis tissues examined, in six breast cancerlines, and 8 melanoma cell lines, and in cultured melanocytes. SEQ IDNO: 3 was found to be expressed in four of the six breast cancer lines,normal testis, liver, kidney, colon and brain. SEQ ID NO: 4 was onlyfound to be expressed by normal testis cells and weakly in brain cells.

EXAMPLE 7

[0038] A further set of experiments were carried out to determine ifantibodies against ING1 were present in sera of normal and cancerpatients. A phase plaque immuno assay of the type described supra wascarried out, using clones of SEQ ID NO: 1 as target. Of 14 allogeneicsera taken from breast cancer patients, two were positive at 1:200dilutions. All normal sera were negative.

EXAMPLE 8

[0039] The BR11 cDNA library described supra was then screened, usingSEQ ID NO: 1 and standard methodologies. A 593 base pair cDNA wasidentified, which was different from any sequences in the data banksconsulted. The sequence of this cDNA molecule is set out at SEQ ID NO:8.

[0040] The cDNA molecule set forth as SEQ ID NO:1 was then used inSouthern blotting experiments. In brief, genomic DNA was isolated fromnormal human tissue, digested with BamHI or Hind III, and then separatedonto 0.7% agarose gel, blotted onto nitrocellulose filters, andhybridized using ³²P labelled SEQ ID NO: 1, at high stringencyconditions (aqueous buffer, 65° C.). The probes were permitted tohybridize overnight, and then exposed for autoradiography. Twohybridizing DNA species were identified, i.e., SEQ ID NOS: 1 and 8.

EXAMPLE 9

[0041] The cDNA molecule set forth in SEQ ID NO: 8 was then analyzed. 5′-RACE PCR was carried out using normal fetus cDNA. Full length cDNA forthe molecule is 771 base pairs long, without the poly A tail. It showsstrong homology to SEQ ID NO: 1, with the strongest homology in the 5′two-thirds (76% identity over nucleotide 1-480); however, the longestORF is only 129 base pairs, and would, encode a poly peptide 42 aminoacids long which was homologous to, but much shorter than, the expectedexpression product of SEQ ID NO: 1.

[0042] In addition to the coding region, SEQ ID NO: 8 contains 203 basepairs of5 ′-untranslated region, and 439 base pairs of 3 ′-untranslatedregion.

[0043] RT-PCR assays were carried out, as described supra. All of thenormal tissues tested, including brain, colon, testis, tissue andbreast, were positive for expression of this gene. Eight melanoma celllines were tested, of which seven showed varying levels of expression,and one showed no expression. Six breast cancer cell lines were tested,of which four showed various levels of expression, and two showed noexpression.

EXAMPLE 10

[0044] An additional breast cancer cDNA library, referred to as“BR17-128”, was screened, using autologous sera. A cDNA molecule wasidentified.

[0045] Analysis of the sequence suggested that it was incomplete at the5′ end. To extend the sequence, a testicular cDNA library was screenedwith a nucleotide probe based upon the partial sequence identified inthe breast cancer library. An additional 1200 base pairs were identifiedfollowing these screenings. The 2011 base pairs of information are setforth in SEQ ID NO: 15.

[0046] The longest open reading frame is 1539 base pairs, correspondingto a protein of about 59.15 kilodaltons. The deduced sequence is setforth at SEQ ID NO: 16.

[0047] RT-PCR was then carried out using the following primers: (SEQ IDNOS: 17 and 18) CACACAGGATCCATGCAGGCCCCGCACAAGGAGCACACAAAGCTTCTAGGATTTGGCACAGCCAGAG

[0048] Strong signals were observed in normal testis and breast tissue,and weak expression was observed in placenta.

[0049] No expression was found in normal brain, kidney, liver, colon,adrenal, fetal brain, lung, pancreas, prostate, thymus, uterus, andovary tissue of tumor cell lines tested, 2 of the breast cancer lineswere strongly positive and two were weakly positive. Of melanoma two of8 were strongly positive, and 3 were weakly positive. Of lung cancercell lines, 4 of 15 were strongly positive, and 3 were weakly positive.

[0050] When cancer tissue specimens were tested, 16 of 25 breast cancersamples were strongly positive, and 3 additional samples were weaklypositive. Two of 36 melanoma samples were positive (one strong, oneweak). All other cancer tissue samples were negative.

[0051] When Northern blotting was carried out, a high molecular weightsmear was observed in testis, but in no other tissues tested.

EXAMPLE 11

[0052] Further experiments were carried out using the tumor samplereferred to in example 10, supra. This sample was derived from asubcutaneous metastasis of a 60 year old female breast cancer patient.Total RNA was extracted, as described supra. Following the extraction, acDNA library was constructed in λ-ZAP expression vectors, also asdescribed supra. Screening was carried out, using the protocol set forthin example 1. A total of 7×10⁵ pfus were screened. Fourteen reactiveclones were identified, purified, and sequenced. The sequences were thencompared to published sequences in GenBank and EST databases. Theseanalyses indicated that the clones were derived from seven distinctgenes, two of which were known, and five unknown. The two known geneswere “PBK-1” (three clones), and TI-227 (one clone). These areuniversally expressed genes, with the libraries referred to suprashowing ESTs for these genes from many different tissues.

[0053] With respect to the remaining 10 clones, six were derived fromthe same gene, referred to hereafter as “NY-BR-1.” Three cDNA sequenceswere found in the EST database which shared identity with the gene. Twoof these (AI 951118 and AW 373574) were identified as being derived froma breast cancer library, while the third (AW 170035), was from a pooledtissue source.

EXAMPLE 12

[0054] The distribution of the new gene NY-BR-1 referred to supra wasdetermined via RT-PCR. In brief, gene specific oligonucleotide NY-BR-1primers were designed to amplify cDNA segments 300-600 base pairs inlength, with primer melting temperatures estimated at 65-70° C.

[0055] The RT-PCR was then carried out over 30 amplification cycles,using a thermal cycler, and an annealing temperature of 60° C. Productswere analyzed via 1.5% gel electrophoresis, and ethidium bromidevisualization. Fifteen normal tissues (adrenal gland, fetal brain, lung,mammary gland, pancreas, placenta, prostate, thymus, uterus, ovary,brain, kidney, liver, colon and testis) were assayed. The NY-BR-1 clonegave a strong signal in mammary gland and testis tissue, and a veryfaint signal in placenta. All other tissues were negative. The otherclones were expressed universally, based upon comparison to informationin the EST database library, and were not pursued further.

[0056] The expression pattern of NY-BR-1 in cancer samples was thentested, by carrying out RT-PCR, as described supra, on tumor samples.

[0057] In order to determine the expression pattern, primers: caaagcagagcctcccgaga ag (SEQ ID NO: 20) and cctatgctgc tcttcgattc ttcc (SEQ ID NO:21)

[0058] Of twenty-five breast cancer samples tested twenty two werepositive for NY-BR-1. Of these, seventeen gave strong signals, and fivegave weak to modest signals.

[0059] An additional 82 non-mammary tumor samples were also analyzed,divided into 36 melanoma, 26 non small cell lung cancer, 6 colon cancer,6 squamous cell carcinoma, 6 transitional cell carcinoma, and twoleiyomyosarcomas. Only two melanoma samples were positive for NY-BR-1expression.

[0060] The study was then extended to expression of NY-BR-1 in tissueculture. Cell lines derived from breast tumor, melanoma, and small celllung cancer were studied. Four of six breast cancer cells were positive(two were very weak), four of eight melanoma (two very weak), and sevenof fourteen small cell lung cancer lines (two very weak) were positive.

EXAMPLE 13

[0061] In order to determine the complete cDNA molecule for NY-BR-1, thesequences of the six clones referred to supra were compiled, to producea nucleotide sequence 1464 base pairs long. Analysis of the open readingframe showed a continuous ORF throughout, indicating that the compiledsequence is not complete.

[0062] Comparison of the compiled sequence with the three EST librarysequences referred to supra allowed for extension of the sequence. TheEST entry AW170035 (446 base pairs long) overlapped the compiledsequence by 89 base pairs at its 5′ end, permitting extension of thesequence by another 357 base pairs. A translational terminal codon wasidentified in this way, leading to a molecule with a 3′-untranslatedregion 333 base pairs long. The 5′ end of the molecule was lacking,however, which led to the experiment described infra.

EXAMPLE 14

[0063] In order to determine the missing, 5′ end of the clone describedsupra, a commercially available testis cDNA expression library wasscreened, using a PCR expression product of the type described supra asa probe. In brief, 5×10⁴ pfus per 150 mm plate were transferred tonitrocellulose membranes, which were then submerged in denaturationsolution (1.5M NaCl and 0.5 M NaOH), transferred to neutralizationsolution (1.5 M NaCl and 0.5M Tris-HCl), and then rinsed with 0.2MTris-HCl, and 2×SSC. Probes were labelled with ³²p and hybridization wascarried out at high stringency conditions (i.e., 68° C., aqueousbuffer). Any positive clones were subcloned, purified, and in vivoexcised to plasmid PBK-CMV, as described supra.

[0064] One of the clones identified in this way included an additional1346 base pairs at the 5′ end; however, it was not a full lengthmolecule. A 5′-RACE-PCR was carried out, using commercially availableproducts. The PCR product was cloned into plasmid vector pGEMT andsequenced. The results indicated that cDNA sequence was extended 1292base pairs further, but no translation initiation site could bedetermined, because no stop codons could be detected. It could beconcluded, however, that the cDNA of the NY-BR17 clone comprises atleast 4026 nucleotides, which are presented as SEQ ID NO: 22. Themolecule, as depicted, encodes a protein at least about 152.8 kDA inmolecular weight. Structurally, there are 99 base pairs 5′ to thepresumed translation initiation site, and an untranslated segment 333base pairs long at the 3′ end. The predicted amino acid sequence of thecoding region for SEQ ID NO: 22 is set out at SEQ. ID NO: 23.

[0065] SEQ ID NO: 23 was analyzed for motifs, using the known searchprograms PROSITE and Pfam. A bipartite nuclear localization signal motifwas identified at amino acids 17-34, suggesting that the protein is anuclear protein. Five tandem ankyrin repeats were identified, at aminoacids 49-81, 82-114, 115-147, 148-180 and 181-213. A bZIP site (i.e. aDNA binding site followed by a leucine zipper motif) was found at aminoacid positions 1077-1104, suggesting a transcription factor function. Itwas also observed that three repetitive elements were identified inbetween the ankyrin repeats and the bZIP DNA binding site. To elaborate,a repetitive element 117 nucleotides long is trandemly repeated 3 times,between amino acids 459-815. The second repetitive sequence, consistingof 11 amino acids, repeats 7 times between amino acids 224 and 300. Thethird repetitive element, 34 amino acids long, is repeated twice,between amino acids 301-368.

EXAMPLE 15

[0066] The six clones described supra were compared, and analysisrevealed that they were derived from two different splice variants.Specifically, two clones, referred to as “BR17-8” and “BR 17-44a”,contain one more exon, of 111 base pairs (nucleotides 3015-3125 of SEQID NO: 22), which encodes amino acids 973-1009 of SEQ ID NO: 23, than doclones BR 17-1a, BR17-35b and BR17-44b. The shortest of the six clones,BR17-128, starts 3′ to the additional exons. The key structural elementsreferred to supra were present in both splice variants, suggesting thatthere was no difference in biological function.

[0067] The expression pattern of the two splice variants was assessedvia PT-PCR, using primers which spanned the 111 base pair exon referredto supra.

[0068] The primers used were: aatgggaaca agagctctgc ag (SEQ ID NO: 24)and gggtcatctg aagttcagca ftc (SEQ ID NO: 25)

[0069] Both variants were expressed strongly in normal testis andbreast. The longer variant was dominant in testis, and the shortervariant in breast cells. When breast cancer cells were tested, co-typingof the variant was observed, (7 strongly, 2 weakly positive, and 1negative), with the shorter variant being the predominant formconsistently.

EXAMPLE 16

[0070] The frequency of antibody response against NY-BR-1 in breastcancer patients was tested. To do this, a recombinant protein consistingof amino acids 993-1188 of SEQ ID NO: 23 was prepared. (This is theprotein encoded by clone BR 17-128, referred to supra). A total of 140serum samples were taken from breast cancer patients, as were 60 normalserum samples. These were analyzed via Western blotting, using standardmethods.

[0071] Four of the cancer sera samples were positive, including a samplefrom patient BR17. All normal sera were negative.

[0072] An additional set of experiments was then carried out todetermine if sera recognized the portion of NY-BR-1 protein withrepetitive elements. To do this, a different recombinant protein,consisting of amino acids 405-1000 was made, and tested in Western blotassays. None of the four antibody positive sera reacted with thisprotein indicating that an antibody epitope is located in thenon-repetitive, carboxy terminal end of the molecule.

EXAMPLE 17

[0073] The screening of the testicular cDNA library referred to supraresulted, inter alia, in the identification of a cDNA molecule that washomologous to NY-BR-1. The molecule is 3673 base pairs in length,excluding the poly A tail. This corresponded to nucleotides 1-3481 ofSEQ ID NO: 22, and showed 62% homology thereto. No sequence identity tosequences in libraries was noted. ORF analysis identified an ORF fromnucleotide 641 through the end of the sequence, with 54% homology to theprotein sequence of SEQ. ID NO: 23. The ACT initiation codon of thissequence is 292 base pairs further 3′ to the presumed initiation codonof NY-BR-1, and is preceded by 640 untranslated base pairs at its 5′end. This 640base pair sequence includes scattered stop codons. Thenucleotide sequence and deduced amino acid sequence are presented as SEQID NOS: 26 and 27, respectively.

[0074] RT-PCR analysis was carried out in the same way as is describedsupra, using primers: tct catagat gctggtgctg atc (SEQ ID NO: 28) andcccagacatt gaattttggc agac. (SEQ ID NO: 29)

[0075] Tissue restricted mRNA expression was found. The expressionpattern differed from that of SEQ ID NO: 22. In brief, of six normaltissues examined, strong signals were found in brain and testis only.There was no or weak expression in normal breast tissues, and kidney,liver and colon tissues were negative. Eight of ten 10 breast cancerspecimens tested supra were positive for SEQ. ID NO: 26. Six sampleswere positive for both SEQ. ID NO: 22 and 26, one for SEQ. ID NO: 22only, two for the SEQ. ID NO: 26 only, and one was negative for both.

EXAMPLE 18

[0076] Recently, a working draft of the human genome sequence wasreleased. This database was searched, using standard methods, andNY-BR-1 was found to have sequence identity with at least threechromosome 10 clones, identified by Genbank accession numbers AL157387,AL37148, and AC067744. These localize NY-BR-1 to chromosome 10p11.21-12.1.

[0077] The comparison of NY-BR-1 and the human genomic sequence led todefinition of NY-BR-1 exon-intron organization. In brief, the codingregion of the gene contains essentially 19 structurally distinct exonswith at least 2 exons encoding 3′ untranslated regions. Detailedexon-intron junction information is described at Genbank AF 269081.

[0078] The six ankyrin repeats, referred to supra, are all found withinexon 7. The 357 nucleotide repeating unit is composed of exons 10-15.The available genomic sequences are not complete, however, and only oneof the three copies was identified, suggesting that DNA sequencesbetween exons 5 and 10 may be duplicated and inserted in tandem, duringgenetic evolution. In brief, when the isolated NY-BR-1 cDNA clone wasanalyzed, three complete and one incomplete copy of the repeating unitsare present. The exon sequences can be expresses as exons1-2-3-4-5-6-7-8-9-(10-11-12-13-14-15)-(10A-11A-12A-13A-14A-15A)-(10B-11B-12B-13B-14B-15B)-(10C-11C-12C-13C-14C)-16-17-18-19-20-21,wherein A, B & C are inexact copies of exon 10-15 sequences. Cloned,NY-BR-1 cDNA has 38 exons in toto.

[0079] It was noted, supra that the sequence of NY-BR-1 cDNA was notcomplete at the 5′ end. Genonic sequence (Genbank AC067744), permittedextension of the 5′ end. Translation of the 5′ genonic sequence led tothe identification of a new translation initiation site, 168 base pairsupstream of the previously predicted ATG initiation codon. This led toan NY-BR-1 polypeptide including 1397 amino acid longer, 56 residue ofwhich are added at the N-terminus, compared to prior sequenceinformation, i.e.:

[0080] MEEISAAAVKVVPGPERPSPFSQLVYTSNDSYIVHSGDLRKIHXAASRGQVRKLEK (SEQ IDNO: 30).

EXAMPLE 20

[0081] Reference was made, supra to the two difference splice variantsof NY-BR-1. Comparison of the splice variants with the genomic sequenceconfirmed that an alternate splicing event, with the longer variantincorporating part of intron 33 into exon 34 (i.e., exon 17 of the basicexon/intron framework described supra).

[0082] Key structural elements that were predicted in NY-BR-1, describedsupra are present in both variants, suggesting that there is nodifference in biological function, or subcellular location.

EXAMPLE 21

[0083] As with NY BR-1, the variant NY-BR-1.1, described supra wasscreened against the working draft of the human genome sequence. Oneclone was found with sequence identity, i.e., GenBank AL359312, derivefrom chromosome 9. Thus, NY-BR-1 and NY-BR-1.1 both appear to befunctioning genes, on two different chromosomes. The Genbank sequencereferred to herein does not contain all of NY-BR-1.1, which precludesdefining exon-intron structure. Nonetheless, at least 3 exons can bedefined, which correspond to exons 16-18 of the NY-BR-1 basic framework.Exon-intron junctions are conserved.

EXAMPLE 22

[0084] A series of peptides were synthesized, based upon the amino acidsequence of NY-BR-1, as set forth in SEQ ID NO: 23. These were thentested for their ability to bind to HLA-A2 molecules and to stimulateCTL proliferation, using an ELISPOT assay. This assay involved coating96-well, flat bottom nitrocellulose plates with 5 ug/ml ofanti-interferon gamma antibodies in 100 ul of PBS per well, followed byovernight incubation. Purified CD8⁺ cells, which had been separated fromPBL samples via magnetic beads coated with anti-CD8 antibodies were thenadded, at 1×10⁵ cells/well, in RPMI 1640 medium, that had beensupplemented with 10% human serum, L-asparagine (50 mg/l), L-arginine(242 mg/l), L-glutamine (300 mg/l), together with IL-2 (2.5 ng/ml), in afinal volume of 100 ul. CD8⁺ effector cells were prepared bypresensitizing with peptide, and were then added at from 5×10³ to 2×10⁴cells/well. Peptides were pulsed onto irradiated T2 cells at aconcentration of 10 ug/ml for 1 hour, washed and added to effectorcells, at 5×10⁴ cells/well. The plates were incubated for 16 hours at37° C., washed six times with 0.05% Tween 20/PBS, and were thensupplemented with biotinylated, anti-interferon gamma specific antibodyat 0.5 ug/ml. After incubation for 2 hours at 37° C., plates werewashed, and developed with commercially available reagents, for 1 hour,followed by 10 minutes of incubation with dye substrate. Plates werethen prepped for counting, positives being indicated by blue spots. Thenumber of blue spots/well was determined as the frequency of NY-ESO-1specific CTLs/well.

[0085] Experiments were run, in triplicate, and total number of CTLs wascalculated. As controls, one of reagents alone, effector cells alone, orantigen presenting cells alone were used. The difference between thenumber of positives in stimulated versus non-stimulated cells, wascalculated as the effective number of peptide specific CTLs abovebackground. Three peptides were found to be reactive, i.e.: (amino acids102-111 of SEQ ID NO: 23) LLSHGAVIEV (amino acids 904-912 of SEQ ID NO:23) SLSKILDTV (amino acids 1262-1270 of SEQ ID NO: 23) SLDQKLFQL.

[0086] The complete list of peptides tested, with reference to theirposition in SEQ ID NO: 23, follows: Peptide Position FLVDRKVCQL 35-43ILIDSGADI 68-76 AVYSEILSV 90-98 ILSVVAKLL  95-103 LLSHGAVIEV 102-111KLLSHGAVI 101-109 FLLIKNANA 134-142 MLLQQNVDV 167-175 GMLLQQNVDV 166-175LLQQNVDVFA 168-177 IAWEKKETPV 361-370 SLFESSAKI 430-438 CIPENSIYQKV441-450 KVMEINREV 449-457 ELMDMQTFKA 687-696 ELMDMQTFKA 806-815SLSKILDTV 904-912 KILDTVHSC 907-915 ILNEKIREEL 987-996 RIQDIELKSV1018-1027 YLLHENCML 1043-1051 CMLKKEIAML 1049-1058 AMLKLELATL 1056-1065KILKEKNAEL 1081-1090 VLIAENTML 1114-1122 CLQRKMNVDV 1174-1183 KMNVDVSST1178-1186 SLDQKLFQL 1262-1270 KLFQLQSKNM 1266-1275 FQLQSKNMWL 1268-1277QLQSKNMWL 1269-1277 NMWLQQQLV 1274-1282 WLQQQLVHA 1276-1284 KITIDIHFL1293-1301

[0087] The foregoing examples describe the isolation of a nucleic acidmolecule which encodes a cancer associated antigen. “Associated” is usedherein because while it is clear that the relevant molecule wasexpressed by several types of cancer, other cancers, not screenedherein, may also express the antigen.

[0088] The invention relates to nucleic acid molecules which encode theantigens encoded by, e.g., SEQ ID NOS: 1, 3, 8, 15, 22 and 26 as well asthe antigens encoded thereby, such as the proteins with the amino acidsequences of SEQ ID NOS: 5, 6, 7, 16, 23, 27, and 30. It is to beunderstood that all sequences which encode the recited antigen are apart of the invention.

[0089] Also apart of the invention are proteins, polypeptides, andpeptides, which comprise, e.g., at least nine consecutive amino acidsfound in SEQ ID NO: 23, or at least nine consecutive amino acids of theamino acids of SEQ ID NO: 30. Proteins, polypeptides and peptidescomprising nine or more amino acids of SEQ ID NO: 5, 6, 7, 16 or 27 arealso a part of the invention. Especially preferred are peptidescomprising or consisting of amino acids 102-111, 904-912, or 1262-1270of SEQ ID NO: 23. Such peptides may, but do not necessarily provoke CTLresponses when complexed with an HLA molecule, such as an HLA-A2molecule. They may also bind to different MHC or HLA molecules,including, but not being limited to, HLA-A1, A2, A3, B7, B8, Cw3, Cw6,or serve, e.g., as immunogens, as part of immunogenic cocktailcompositions, where they are combined with other proteins orpolypeptides, and so forth. Also a part of the invention are the nucleicacid molecules which encode these molecules, such as “minigenes,”expression vectors that include the coding regions, recombinant cellscontaining these, and so forth. All are a part of the invention.

[0090] Also a part of the invention are expression vectors whichincorporate the nucleic acid molecules of the invention, in operablelinkage (i.e., “operably linked”) to a promoter. Construction of suchvectors, such as viral (e.g., adenovirus or Vaccinia virus) orattenuated viral vectors is well within the skill of the art, as is thetransformation or transfection of cells, to produce eukaryotic celllines, or prokaryotic cell strains which encode the molecule ofinterest. Exemplary of the host cells which can be employed in thisfashion are COS cells, CHO cells, yeast cells, insect cells (e.g.,Snodoptera frugiperda), NIH 3T3 cells, and so forth. Prokaryotic cells,such as E. coil and other bacteria may also be used. Any of these cellscan also be transformed or transfected with further nucleic acidmolecules, such as those encoding cytokines, e.g., interleukins such asIL-2, 4, 6, or 12 or HLA or MHC molecules.

[0091] Also a part of the invention are the antigens described herein,both in original form and in any different post translational modifiedforms. The molecules are large enough to be antigenic without anyposttranslational modification, and hence are useful as immunogens, whencombined with an adjuvant (or without it), in both precursor andpost-translationally modified forms. Antibodies produced using theseantigens, both poly and monoclonal, are also a part of the invention aswell as hybridomas which make monoclonal antibodies to the antigens. Thewhole protein can be used therapeutically, or in portions, as discussedinfra. Also a part of the invention are antibodies against this antigen,be these polyclonal, monoclonal, reactive fragments, such as Fab,(F(ab)₂′ and other fragments, as well as chimeras, humanized antibodies,recombinantly produced antibodies, and so forth.

[0092] As is clear from the disclosure, one may use the proteins andnucleic acid molecules of the invention diagnostically. The SEREXmethodology discussed herein is premised on an immune response to apathology associated antigen. Hence, one may assay for the relevantpathology via, e.g., testing a body fluid sample of a subject, such asserum, for reactivity with the antigen per se. Reactivity would bedeemed indicative of possible presence of the pathology. So, too, couldone assay for the expression of any of the antigens via any of thestandard nucleic acid hybridization assays which are well known to theart, and need not be elaborated upon herein. One could assay forantibodies against the subject molecules, using standard immunoassays aswell.

[0093] Analysis of SEQ ID NO: 1, 3, 4, 8, 15, 22 and 26 will show thatthere are 5′ and 3′ non-coding regions presented therein. The inventionrelates to those isolated nucleic acid molecules which contain at leastthe coding segment, and which may contain any or all of the non-coding5′ and 3′ portions.

[0094] Also apart of the invention are portions of the relevant nucleicacid molecules which can be used, for example, as oligonucleotideprimers and/or probes, such as one or more of SEQ ID NOS: 9, 10, 11, 12,13, 14, 17, 18, 20, 21, 24, 25, 28, and 29 as well as amplificationproducts like nucleic acid molecules comprising at least nucleotides305-748 of SEQ ID NO: 1, or amplification products described in theexamples, including those in examples 12, 14, etc.

[0095] As was discussed supra, study of other members of the “CT” familyreveals that these are also processed to peptides which provoke lysis bycytolytic T cells, There has been a great deal of work on motifs forvarious MHC or HLA molecules, which is applicable here. Hence, a furtheraspect of the invention is a therapeutic method, wherein one or morepeptides derived from the antigens of the invention which bind to an HLAmolecule on the surface of a patient's tumor cells are administered tothe patient, in an amount sufficient for the peptides to bind to theMHC/HLA molecules, and provoke lysis by T cells. Any combination ofpeptides may be used. These peptides, which may be used alone or incombination, as well as the entire protein or immunoreactive portionsthereof, may be administered to a subject in need thereof, using any ofthe standard types of administration, such as intravenous, intradermal,subcutaneous, oral, rectal, and transdermal administration. Standardpharmaceutical carriers, adjuvants, such as saponins, GM-CSF, andinterleukins and so forth may also be used. Further, these peptides andproteins may be formulated into vaccines with the listed material, asmay dendritic cells, or other cells which present relevant MHC/peptidecomplexes.

[0096] Similarly, the invention contemplates therapies wherein nucleicacid molecules which encode the proteins of the invention, one or moreor peptides which are derived from these proteins are incorporated intoa vector, such as a Vaccinia or adenovirus based vector, to render ittransfectable into eukaryotic cells, such as human cells. Similarly,nucleic acid molecules which encode one or more of the peptides may beincorporated into these vectors, which are then the major constituent ofnucleic acid bases therapies.

[0097] Any of these assays can also be used in progression/regressionstudies. One can monitor the course of abnormality involving expressionof these antigens simply by monitoring levels of the protein, itsexpression, antibodies against it and so forth using any or all of themethods set forth supra.

[0098] It should be clear that these methodologies may also be used totrack the efficacy of a therapeutic regime. Essentially, one can take abaseline value for a protein of interest using any of the assaysdiscussed supra, administer a given therapeutic agent, and then monitorlevels of the protein thereafter, observing changes in antigen levels asindicia of the efficacy of the regime.

[0099] As was indicated supra, the invention involves, inter alias therecognition of an “integrated” immune response to the molecules of theinvention. One ramification of this is the ability to monitor the courseof cancer therapy. In this method, which is a part of the invention, asubject in need of the therapy receives a vaccination of a typedescribed herein. Such a vaccination results, e.g., in a T cell responseagainst cells presenting HLA/peptide complexes on their cells. Theresponse also includes an antibody response, possibly a result of therelease of antibody provoking proteins via the lysis of cells by the Tcells. Hence, one can monitor the effect of a vaccine, by monitoring anantibody response. As is indicated, supra, an increase in antibody titermay be taken as an indicia of progress with a vaccine, and vice versa.Hence, a further aspect of the invention is a method for monitoringefficacy of a vaccine, following administration thereof, by determininglevels of antibodies in the subject which are specific for the vaccineitself, or a large molecule of which the vaccine is a part.

[0100] The identification of the subject proteins as being implicated inpathological conditions such as cancer also suggests a number oftherapeutic approaches in addition to those discussed supra. Theexperiments set forth supra establish that antibodies are produced inresponse to expression of the protein. Hence, a further embodiment ofthe invention is the treatment of conditions which are characterized byaberrant or abnormal levels of one or more of the proteins, viaadministration of antibodies, such as humanized antibodies, antibodyfragments, and so forth. These may be tagged or labelled withappropriate cystostatic or cytotoxic reagents.

[0101] T cells may also be administered. It is to be noted that the Tcells maybe elicited in vitro using immune responsive cells such asdendritic cells, lymphocytes, or any other immune responsive cells, andthen reperfused into the subject being treated.

[0102] Note that the generation of T cells and/or antibodies can also beaccomplished by administering cells, preferably treated to be renderednon-proliferative, which present relevant T cell or B cell epitopes forresponse, such as the epitopes discussed supra.

[0103] The therapeutic approaches may also include antisense therapies,wherein an antisense molecule, preferably from 10 to 100 nucleotides inlength, is administered to the subject either “neat” or in a carrier,such as a liposome, to facilitate incorporation into a cell, followed byinhibition of expression of the protein. Such antisense sequences mayalso be incorporated into appropriate vaccines, such as in viral vectors(e.g., Vaccinia), bacterial constructs, such as variants of the knownBCG vaccine, and so forth.

[0104] Other features and applications of the invention will be clear tothe skilled artisan, and need not be set forth herein. The terms andexpression which have been employed are used as terms of description andnot of limitation, and there is no intention in the use of such termsand expression of excluding any equivalents of the features shown anddescribed or portions thereof, it being recognized that variousmodifications are possible within the scope of the invention.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 19 <210> SEQ ID NO 1<211> LENGTH: 1533 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: 235 <223> OTHERINFORMATION: unknown <400> SEQUENCE: 1 ggttttccac gttggacaag tgcggctcggcggccagcgg agcgcgcccc ttcccgctgc 60 ccgctccgct cctctcttct acccagcccagtgggcgagt gggcagcggc ggccgcggcg 120 ctgggccctc tcccgccggt gtgtgcgcgctcgtacgcgc ggcccccggc gccagccccg 180 ccgcctgaga gggggcctgc gccgccggccggggcgtgcg cccgggagcc accgncaccg 240 cggcccgcgc cctcaggcgc tggggtccccgcggacccgg aggcggcgga cgggctcggc 300 agatgtagcc gccgggccga agcaggagccggcggggggg cgccgggaga gcgagggctt 360 tgcattttgc agtgctattt tttgaggggggcggagggtg gaggaagtcg gaaagccgcg 420 ccgagtcgcc ggggacctcc ggggtgaaccatgttgagtc ctgccaacgg ggagcagctc 480 cacctggtga actatgtgga ggactacctggactccatcg agtccctgcc tttcgacttg 540 cagagaaatg tctcgctgat gcgggagatcgacgcgaaat accaagagat cctgaaggag 600 ctagacgagt gctacgagcg cttcagtcgcgagacagacg gggcgcagaa gcggcggatg 660 ctgcactgtg tgcagcgcgc gctgatccgcagccaggagc tgggcgacga gaagatccag 720 atcgtgagcc agatggtgga gctggtggagaaccgcacgc ggcaggtgga cagccacgtg 780 gagctgttcg aggcgcagca ggagctgggcgacacagcgg gcaacagcgg caaggctggc 840 gcggacaggc ccaaaggcga ggcggcagcgcaggctgaca agcccaacag caagcgctca 900 cggcggcagc gcaacaacga gaaccgtgagaacgcgtcca gcaaccacga ccacgacgac 960 ggcgcctcgg gcacacccaa ggagaagaaggccaagacct ccaagaagaa gaagcgctcc 1020 aaggccaagg cggagcgaga ggcgtcccctgccgacctcc ccatcgaccc caacgaaccc 1080 acgtactgtc tgtgcaacca ggtctcctatggggagatga tcggctgcga caacgacgag 1140 tgccccatcg agtggttcca cttctcgtgcgtggggctca atcataaacc caagggcaag 1200 tggtactgtc ccaagtgccg gggggagaacgagaagacca tggacaaagc cctggagaaa 1260 tccaaaaaag agagggctta caacaggtagtttgtggaca ggcgcctggt gtgaggagga 1320 caaaataaac cgtgtattta ttacattgctgcctttgttg aggtgcaagg agtgtaaaat 1380 gtatattttt aaagaatgtt agaaaaggaaccattccttt catagggatg gcagtgattc 1440 tgtttgcctt ttgttttcat tggtacacgtgtaacaagaa agtggtctgt ggatcagcat 1500 tttagaaact acaaatatag gtttgattcaaca 1533 <210> SEQ ID NO 2 <211> LENGTH: 1143 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <400> SEQUENCE: 2 gagtaacccg ataatatgccgttgtccggc acggcgacga gaattcccag atatagcagt 60 agcagtgatc ccgggcctgtggctcggggc cggggctgca gttcggaccg cctcccgcga 120 cccgcggggg ctcggagacagtttcaggcc gcatctttgc tgacccgagg gtggggccgc 180 gcgtggccgt ggaaacagatcctgaaggag ctagacgagt gctacgagcg cttcagtcgc 240 gagacagacg gggcgcagaagcggcggatg ctgcactgtg tgcagcgcgc gctgatccgc 300 agccaggagc tgggcgacgagaagatccag atcgtgagcc agatggtgga gctggtggag 360 aaccgcacgc ggcaggtggacagccacgtg gagctgttcg aggcgcagca ggagctgggc 420 gacacagtgg gcaacagcggcaaggttggc gcggacaggc ccaatggcga tgcggtagcg 480 cagtctgaca agcccaacagcaagcgctca cggcggcagc gcaacaacga gaaccgtgag 540 aacgcgtcca gcaaccacgaccacgacgac ggcgcctcgg gcacacccaa ggagaagaag 600 gccaagacct ccaagaagaagaagcgctcc aaggccaagg cggagcgaga ggcgtcccct 660 gccgacctcc ccatcgaccccaacgaaccc acgtactgtc tgtgcaacca ggtctcctat 720 ggggagatga tcggctgcgacaacgacgag tgccccatcg agtggttcca cttctcgtgc 780 gtggggctca atcataaacccaagggcaag tggtactgtc ccaagtgccg gggggagaac 840 gagaagacca tggacaaagccctggagaaa tccaaaaaag agagggctta caacaggtag 900 tttgtggaca ggcgcctggtgtgaggagga caaaataaac cgtgtattta ttacattgct 960 gcctttgttg aggtgcaaggagtgtaaaat gtatattttt aaagaatgtt agaaaaggaa 1020 ccattccttt catagggatggcagtgattc tgtttgcctt ttgttttcat tggtacacgt 1080 gtaacaagaa agtggtctgtggatcagcat tttagaaact acaaatatag gtttgattca 1140 aca 1143 <210> SEQ IDNO 3 <211> LENGTH: 742 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <400> SEQUENCE: 3 cgccgtccac accccagcgg ccctgacgctgtcccctccg cgaccctcgc ctctggaaaa 60 agtgacaggc aaggccacgc ccccgcgagggccggcctcg agcccgcagc ccccagggcc 120 tgggacgaga tcctgaagga gctagacgagtgctacgagc gcttcagtcg cgagacagac 180 ggggcgcaga agcggcggat gctgcactgtgtgcagcgcg cgctgatccg cagccaggag 240 ctgggcgacg agaagatcca gatcgtgagccagatggtgg agctggtgga gaaccgcacg 300 cggcaggtgg acagccacgt ggagctgttcgaggcgcagc aggagctggg cgacacagcg 360 ggcaacagcg gcaaggctgg cgcggacaggcccaaaggcg aggcggcagc gcaggctgac 420 aagcccaaca gcaagcgctc acggcggcagcgcaacaacg agaaccgtga gaacgcgtcc 480 agcaaccacg accacgacga cggcgcctcgggcacaccca aggagaagaa ggccaagacc 540 tccaagaaga agaagcgctc caaggccaaggcggagcgag aggcgtcccc tgccgacctc 600 cccatcgacc ccaacgaacc cacgtactgtctgtgcaacc aggtctccta tggggagatg 660 atcggctgcg acaacgacga gtgccccatcgagtggttcc acttctcgtg cgtggggctc 720 aatcataaac ccaagggcaa gt 742 <210>SEQ ID NO 4 <211> LENGTH: 857 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 4 cctccgagaa cggtgtccat ggcacagggc gggaagagataaggcctagg gaaggcgccc 60 ctcgggccta tccacctctt ctggggctcg gcactaggaagcagcttccc tctcaggccc 120 ctttgtctcc aagccgttcc aaactgagta ccgggagacgacacaaaggg agggcggtga 180 cggatggcgc aggcgcggga gccgcctagg ctgctgggagtggtggtccg gccgcggaat 240 ggagatcctg aaggagctag acgagtgcta cgagcgcttcagtcgcgaga cagacggggc 300 gcagaagcgg cggatgctgc actgtgtgca gcgcgcgctgatccgcagcc aggagctggg 360 cgacgagaag atccagatcg tgagccagat ggtggagctggtggagaacc gcacgcggca 420 ggtggacagc cacgtggagc tgttcgaggc gcagcaggagctgggcgaca cagcgggcaa 480 cagcggcaag gctggcgcgg acaggcccaa aggcgaggcggcagcgcagg ctgacaagcc 540 caacagcaag cgctcacggc ggcagcgcaa caacgagaaccgtgagaacg cgtccagcaa 600 ccacgaccac gacgacggcg cctcgggcac acccaaggagaagaaggcca agacctccaa 660 gaagaagaag cgctccaagg ccaaggcgga gcgagaggcgtcccctgccg acctccccat 720 cgaccccaac gaacccacgt actgtctgtg caaccaggtctcctatgggg agatgatcgg 780 ctgcgacaac gacgagtgcc ccatcgagtg gttccacttctcgtgcgtgg ggctcaatca 840 taaacccaag ggcaagt 857 <210> SEQ ID NO 5 <211>LENGTH: 279 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:5 Met Leu Ser Pro Ala Asn Gly Glu Gln Leu His Leu Val Asn Tyr Val 1 5 1015 Glu Asp Tyr Leu Asp Ser Ile Glu Ser Leu Pro Phe Asp Leu Gln Arg 20 2530 Asn Val Ser Leu Met Arg Glu Ile Asp Ala Lys Tyr Gln Glu Ile Leu 35 4045 Lys Glu Leu Asp Glu Cys Tyr Glu Arg Phe Ser Arg Glu Thr Asp Gly 50 5560 Ala Gln Lys Arg Arg Met Leu His Cys Val Gln Arg Ala Leu Ile Arg 65 7075 80 Ser Gln Glu Leu Gly Asp Glu Lys Ile Gln Ile Val Ser Gln Met Val 8590 95 Glu Leu Val Glu Asn Arg Thr Arg Gln Val Asp Ser His Val Glu Leu100 105 110 Phe Glu Ala Gln Gln Glu Leu Gly Asp Thr Val Gly Asn Ser GlyLys 115 120 125 Val Gly Ala Asp Arg Pro Asn Gly Asp Ala Val Ala Gln SerAsp Lys 130 135 140 Pro Asn Ser Lys Arg Ser Arg Arg Gln Arg Asn Asn GluAsn Arg Glu 145 150 155 160 Asn Ala Ser Ser Asn His Asp His Asp Asp GlyAla Ser Gly Thr Pro 165 170 175 Lys Glu Lys Lys Ala Lys Thr Ser Lys LysLys Lys Arg Ser Lys Ala 180 185 190 Lys Ala Glu Arg Glu Ala Ser Pro AlaAsp Leu Pro Ile Asp Pro Asn 195 200 205 Glu Pro Thr Tyr Cys Leu Cys AsnGln Val Ser Tyr Gly Glu Met Ile 210 215 220 Gly Cys Asp Asn Asp Glu CysPro Ile Glu Trp Phe His Phe Ser Cys 225 230 235 240 Val Gly Leu Asn HisLys Pro Lys Gly Lys Trp Tyr Cys Pro Lys Cys 245 250 255 Arg Gly Glu AsnGlu Lys Thr Met Asp Lys Ala Leu Glu Lys Ser Lys 260 265 270 Lys Glu ArgAla Tyr Asn Arg 275 <210> SEQ ID NO 6 <211> LENGTH: 210 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <220> FEATURE: <400> SEQUENCE: 6 Met LeuHis Cys Val Gln Arg Ala Leu Ile Arg Ser Gln Glu Leu Gly 1 5 10 15 AspGlu Lys Ile Gln Ile Val Ser Gln Met Val Glu Leu Val Glu Asn 20 25 30 ArgThr Arg Gln Val Asp Ser His Val Glu Leu Phe Glu Ala Gln Gln 35 40 45 GluLeu Gly Asp Thr Val Gly Asn Ser Gly Lys Val Gly Ala Asp Arg 50 55 60 ProAsn Gly Asp Ala Val Ala Gln Ser Asp Lys Pro Asn Ser Lys Arg 65 70 75 80Ser Arg Arg Gln Arg Asn Asn Glu Asn Arg Glu Asn Ala Ser Ser Asn 85 90 95His Asp His Asp Asp Gly Ala Ser Gly Thr Pro Lys Glu Lys Lys Ala 100 105110 Lys Thr Ser Lys Lys Lys Lys Arg Ser Lys Ala Lys Ala Glu Arg Glu 115120 125 Ala Ser Pro Ala Asp Leu Pro Ile Asp Pro Asn Glu Pro Thr Tyr Cys130 135 140 Leu Cys Asn Gln Val Ser Tyr Gly Glu Met Ile Gly Cys Asp AsnAsp 145 150 155 160 Glu Cys Pro Ile Glu Trp Phe His Phe Ser Cys Val GlyLeu Asn His 165 170 175 Lys Pro Lys Gly Lys Trp Tyr Cys Pro Lys Cys ArgGly Glu Asn Glu 180 185 190 Lys Thr Met Asp Lys Ala Leu Glu Lys Ser LysLys Glu Arg Ala Tyr 195 200 205 Asn Arg 210 <210> SEQ ID NO 7 <211>LENGTH: 235 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:7 Met Glu Ile Leu Lys Glu Leu Asp Glu Cys Tyr Glu Arg Phe Ser Arg 1 5 1015 Glu Thr Asp Gly Ala Gln Lys Arg Arg Met Leu His Cys Val Gln Arg 20 2530 Ala Leu Ile Arg Ser Gln Glu Leu Gly Asp Glu Lys Ile Gln Ile Val 35 4045 Ser Gln Met Val Glu Leu Val Glu Asn Arg Thr Arg Gln Val Asp Ser 50 5560 His Val Glu Leu Phe Glu Ala Gln Gln Glu Leu Gly Asp Thr Val Gly 65 7075 80 Asn Ser Gly Lys Val Gly Ala Asp Arg Pro Asn Gly Asp Ala Val Ala 8590 95 Gln Ser Asp Lys Pro Asn Ser Lys Arg Ser Arg Arg Gln Arg Asn Asn100 105 110 Glu Asn Arg Glu Asn Ala Ser Ser Asn His Asp His Asp Asp GlyAla 115 120 125 Ser Gly Thr Pro Lys Glu Lys Lys Ala Lys Thr Ser Lys LysLys Lys 130 135 140 Arg Ser Lys Ala Lys Ala Glu Arg Glu Ala Ser Pro AlaAsp Leu Pro 145 150 155 160 Ile Asp Pro Asn Glu Pro Thr Tyr Cys Leu CysAsn Gln Val Ser Tyr 165 170 175 Gly Glu Met Ile Gly Cys Asp Asn Asp GluCys Pro Ile Glu Trp Phe 180 185 190 His Phe Ser Cys Val Gly Leu Asn HisLys Pro Lys Gly Lys Trp Tyr 195 200 205 Cys Pro Lys Cys Arg Gly Glu AsnGlu Lys Thr Met Asp Lys Ala Leu 210 215 220 Glu Lys Ser Lys Lys Glu ArgAla Tyr Asn Arg 225 230 235 <210> SEQ ID NO 8 <211> LENGTH: 772 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: 689,714 <223> OTHER INFORMATION: Unknown <400>SEQUENCE: 8 aaagcgttct cggcggcagc gcaacaacta gaaccgtgag aacgcgtccagcaaccgcga 60 cccacgacga cgtcacctcg ggcacgccca aggagaagaa agcccagacctctaagaaga 120 agcagggctc catggccaag gcgtagcggc aggcgtcccc cgcagacctccccatcgacc 180 ccagcgagcc ctcctactgg gagatgatcc gctgcgacaa cgaatgccccatcgagtggt 240 tccgcttctc gtgtgtgagt ctcaaccata aaccaaagcg caagtggtactgttccagat 300 gccggggaaa gaacgatggg caaagccctt gagaagtcca gaaaaaaaacagggcttata 360 acaggtagtt tggggacatg cgtctaatag tgaggagaac aaaataagccagtgtgttga 420 ttacattgcc acctttgctg aggtgcagga agtgtaaaat gtatatttttaaagaatgtt 480 gttagaggcc gggcgcggtg gctcacgcct gtaatcccag cactttgggaggccgaggcg 540 gtcggatcac gaggtcagga gatcgagacc atcctggcta acacggtgaaaccccgtctc 600 tactaaaaat tcaaaaaaaa aattagctgg gcgtggtggc gggcgcctgtagtcccagct 660 attcgggagg ctgaggcagg agaatggcnt gaacctggga ggtggagcttgcantgagcc 720 aaggtcgcgc cactgcactc cagcctgggc gacagagcga gactccatct ta772 <210> SEQ ID NO 9 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <400> SEQUENCE: 9 cacacaggat ccatgttgag tcctgccaac gg 32<210> SEQ ID NO 10 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 10 cgtggtcgtg gttgctggac gcg 23 <210> SEQ ID NO11 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 11 cccagcggcc ctgacgctgt t 21 <210> SEQ ID NO 12 <211> LENGTH:23 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 12cgtggtcgtg gttgctggac gcg 23 <210> SEQ ID NO 13 <211> LENGTH: 23 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 13 ggaagagataaggcctaggg aag 23 <210> SEQ ID NO 14 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 14 cgtggtcgtg gttgctggacgcg 23 <210> SEQ ID NO 15 <211> LENGTH: 2030 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: 1628, 1752, 1758, 1769, 1789, 1873, 1908, 1915, 1933, 1970,1976,2022 <223> OTHER INFORMATION: Unknown <400> SEQUENCE: 15 ctcgtgccgttaaagatggt cttctgaagg ctaactgcgg aatgaaagtt tctattccaa 60 ctaaagccttagaattgatg gacatgcaaa ctttcaaagc agagcctccc gagaagccat 120 ctgccttcgagcctgccatt gaaatgcaaa agtctgttcc aaataaagcc ttggaattga 180 agaatgaacaaacattgaga gcagatgaga tactcccatc agaatccaaa caaaaggact 240 atgaagaaagttcttgggat tctgagagtc tctgtgagac tgtttcacag aaggatgtgt 300 gtttacccaaggctacacat caaaaagaaa tagataaaat aaatggaaaa ttagaagagt 360 ctcctgataatgatggtttt ctgaaggctc cctgcagaat gaaagtttct attccaacta 420 aagccttagaattgatggac atgcaaactt tcaaagcaga gcctcccgag aagccatctg 480 ccttcgagcctgccattgaa atgcaaaagt ctgttccaaa taaagccttg gaattgaaga 540 atgaacaaacattgagagca gatcagatgt tcccttcaga atcaaaacaa aagaaggttg 600 aagaaaattcttgggattct gagagtctcc gtgagactgt ttcacagaag gatgtgtgtg 660 tacccaaggctacacatcaa aaagaaatgg ataaaataag tggaaaatta gaagattcaa 720 ctagcctatcaaaaatcttg gatacagttc attcttgtga aagagcaagg gaacttcaaa 780 aagatcactgtgaacaacgt acaggaaaaa tggaacaaat gaaaaagaag ttttgtgtac 840 tgaaaaagaaactgtcagaa gcaaaagaaa taaaatcaca gttagagaac caaaaagtta 900 aatgggaacaagagctctgc agtgtgagat tgactttaaa ccaagaagaa gagaagagaa 960 gaaatgccgatatattaaat gaaaaaatta gggaagaatt aggaagaatc gaagagcagc 1020 ataggaaagagttagaagtg aaacaacaac ttgaacaggc tctcagaata caagatatag 1080 aattgaagagtgtagaaagt aatttgaatc aggtttctca cactcatgaa aatgaaaatt 1140 atctcttacatgaaaattgc atgttgaaaa aggaaattgc catgctaaaa ctggaaatag 1200 ccacactgaaacaccaatac caggaaaagg aaaataaata ctttgaggac attaagattt 1260 taaaagaaaagaatgctgaa cttcagatga ccctaaaact gaaagaggaa tcattaacta 1320 aaagggcatctcaatatagt gggcagctta aagttctgat agctgagaac acaatgctca 1380 cttctaaattgaaggaaaaa caagacaaag aaatactaga ggcagaaatt gaatcacacc 1440 atcctagactggcttctgct gtacaagacc atgatcaaat tgtgacatca agaaaaagtc 1500 aagaacctgctttccacatt gcaggagatg cttgtttgca aagaaaaatg aatgttgatg 1560 tgagtagtaccgatatataa caatgaggtg ctccatcaac cactttctga agctcaaagg 1620 aaatccanaagcctaaaaat taatctcaat tatgcaggag atgctctaag agaaaataca 1680 ttggtttcaggaacatgcac aaagagacca acgtgaaaca cagtgtcaaa tgaaggaagc 1740 tgaacacatgtntcaaancg aacaagatna tgtgaacaaa cacactganc agcaggagtc 1800 tctagatcagaaattatttc aactacaaag caaaaatatg tggcttcaac agcaattagt 1860 tcatgcacataangaaagct gacaacaaaa gcaagataac aattgatntt cattntcttg 1920 agaggaaaatgcncatcatc ttctaaaaga gaaaaatgag gagatatttn attacnataa 1980 ccatttaaaaaacccgtata tttcaatatg gaaaaaaaaa anaaaaaaaa 2030 <210> SEQ ID NO 16<211> LENGTH: 512 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 16 Met Lys Val Ser Ile Pro Thr Lys Ala Leu Glu Leu Met Asp MetGln 1 5 10 15 Thr Phe Lys Ala Glu Pro Pro Glu Lys Pro Ser Ala Phe GluPro Ala 20 25 30 Ile Glu Met Gln Lys Ser Val Pro Asn Lys Ala Leu Glu LeuLys Asn 35 40 45 Glu Gln Thr Leu Arg Ala Asp Glu Ile Leu Pro Ser Glu SerLys Gln 50 55 60 Lys Asp Tyr Glu Glu Ser Ser Trp Asp Ser Glu Ser Leu CysGlu Thr 65 70 75 80 Val Ser Gln Lys Asp Val Cys Leu Pro Lys Ala Thr HisGln Lys Glu 85 90 95 Ile Asp Lys Ile Asn Gly Lys Leu Glu Glu Ser Pro AspAsn Asp Gly 100 105 110 Phe Leu Lys Ala Pro Cys Arg Met Lys Val Ser IlePro Thr Lys Ala 115 120 125 Leu Glu Leu Met Asp Met Gln Thr Phe Lys AlaGlu Pro Pro Glu Lys 130 135 140 Pro Ser Ala Phe Glu Pro Ala Ile Glu MetGln Lys Ser Val Pro Asn 145 150 155 160 Lys Ala Leu Glu Leu Lys Asn GluGln Thr Leu Arg Ala Asp Gln Met 165 170 175 Phe Pro Ser Glu Ser Lys GlnLys Lys Val Glu Glu Asn Ser Trp Asp 180 185 190 Ser Glu Ser Leu Arg GluThr Val Ser Gln Lys Asp Val Cys Val Pro 195 200 205 Lys Ala Thr His GlnLys Glu Met Asp Lys Ile Ser Gly Lys Leu Glu 210 215 220 Asp Ser Thr SerLeu Ser Lys Ile Leu Asp Thr Val His Ser Cys Glu 225 230 235 240 Arg AlaArg Glu Leu Gln Lys Asp His Cys Glu Gln Arg Thr Gly Lys 245 250 255 MetGlu Gln Met Lys Lys Lys Phe Cys Val Leu Lys Lys Lys Leu Ser 260 265 270Glu Ala Lys Glu Ile Lys Ser Gln Leu Glu Asn Gln Lys Val Lys Trp 275 280285 Glu Gln Glu Leu Cys Ser Val Arg Leu Thr Leu Asn Gln Glu Glu Glu 290295 300 Lys Arg Arg Asn Ala Asp Ile Leu Asn Glu Lys Ile Arg Glu Glu Leu305 310 315 320 Gly Arg Ile Glu Glu Gln His Arg Lys Glu Leu Glu Val LysGln Gln 325 330 335 Leu Glu Gln Ala Leu Arg Ile Gln Asp Ile Glu Leu LysSer Val Glu 340 345 350 Ser Asn Leu Asn Gln Val Ser His Thr His Glu AsnGlu Asn Tyr Leu 355 360 365 Leu His Glu Asn Cys Met Leu Lys Lys Glu IleAla Met Leu Lys Leu 370 375 380 Glu Ile Ala Thr Leu Lys His Gln Tyr GlnGlu Lys Glu Asn Lys Tyr 385 390 395 400 Phe Glu Asp Ile Lys Ile Leu LysGlu Lys Asn Ala Glu Leu Gln Met 405 410 415 Thr Leu Lys Leu Lys Glu GluSer Leu Thr Lys Arg Ala Ser Gln Tyr 420 425 430 Ser Gly Gln Leu Lys ValLeu Ile Ala Glu Asn Thr Met Leu Thr Ser 435 440 445 Lys Leu Lys Glu LysGln Asp Lys Glu Ile Leu Glu Ala Glu Ile Glu 450 455 460 Ser His His ProArg Leu Ala Ser Ala Val Gln Asp His Asp Gln Ile 465 470 475 480 Val ThrSer Arg Lys Ser Gln Glu Pro Ala Phe His Ile Ala Gly Asp 485 490 495 AlaCys Leu Gln Arg Lys Met Asn Val Asp Val Ser Ser Thr Asp Ile 500 505 510<210> SEQ ID NO 17 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 17 cacacaggat ccatgcaggc cccgcacaag gag 33 <210>SEQ ID NO 18 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 18 cacacaaagc ttctaggatt tggcacagcc agag 34<210> SEQ ID NO 19 <211> LENGTH: 294 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <400> SEQUENCE: 19 Met Pro Leu Cys Thr Ala Thr Arg Ile ProArg Tyr Ser Ser Ser Ser 1 5 10 15 Asp Pro Gly Pro Val Ala Arg Gly ArgGly Cys Ser Ser Asp Arg Leu 20 25 30 Pro Arg Pro Ala Gly Pro Ala Arg ArgGln Phe Gln Ala Ala Ser Leu 35 40 45 Leu Thr Arg Gly Trp Gly Arg Ala TrpPro Trp Lys Gln Ile Leu Lys 50 55 60 Glu Leu Asp Glu Cys Tyr Glu Arg PheSer Arg Glu Thr Asp Gly Ala 65 70 75 80 Gln Lys Arg Arg Met Leu His CysVal Gln Arg Ala Leu Ile Arg Ser 85 90 95 Gln Glu Leu Gly Asp Glu Lys IleGln Ile Val Ser Gln Met Val Glu 100 105 110 Leu Val Glu Asn Arg Thr ArgGln Val Asp Ser His Val Glu Leu Phe 115 120 125 Glu Ala Gln Gln Glu LeuGly Asp Thr Val Gly Asn Ser Gly Lys Val 130 135 140 Gly Ala Asp Arg ProAsn Gly Asp Ala Val Ala Gln Ser Asp Lys Pro 145 150 155 160 Asn Ser LysArg Ser Arg Arg Gln Arg Asn Asn Glu Asn Arg Glu Asn 165 170 175 Ala SerSer Asn His Asp His Asp Asp Gly Ala Ser Gly Thr Pro Lys 180 185 190 GluLys Lys Ala Lys Thr Ser Lys Lys Lys Lys Arg Ser Lys Ala Lys 195 200 205Ala Glu Arg Glu Ala Ser Pro Ala Asp Leu Pro Ile Asp Pro Asn Glu 210 215220 Pro Thr Tyr Cys Leu Cys Asn Gln Val Ser Tyr Gly Glu Met Ile Gly 225230 235 240 Cys Asp Asn Asp Glu Cys Pro Ile Glu Trp Phe His Phe Ser CysVal 245 250 255 Gly Leu Asn His Lys Pro Lys Gly Lys Trp Tyr Cys Pro LysCys Arg 260 265 270 Gly Glu Asn Glu Lys Thr Met Asp Lys Ala Leu Glu LysSer Lys Lys 275 280 285 Glu Arg Ala Tyr Asn Arg 290 294

We claim:
 1. Isolated nucleic acid molecule which encodes a cancerassociated antigen, whose amino acid sequence is identical to the aminoacid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, 3, 4,8, 15, 19, 22, or
 26. 2. The isolated nucleic acid molecule of claim 1,comprising the nucleotide sequence of SEQ ID NO:
 1. 3. The isolatednucleic acid molecule of claim 1, comprising the nucleotide sequence ofSEQ ID NO:
 3. 4. The isolated nucleic acid molecule of claim 1,comprising the nucleotide sequence of SEQ ID NO:
 4. 5. The isolatednucleic acid molecule of claim 1, comprising the nucleotide sequence ofSEQ ID NO:
 8. 6. The isolated nucleic acid molecule of claim 1,comprising the nucleotide sequence of SEQ ID NO:
 15. 7. The isolatednucleic acid molecule of claim 1, comprising the nucleotide sequence ofSEQ ID NO:
 19. 8. The isolated nucleic acid molecule of claim 1,comprising the nucleotide sequence of SEQ ID NO:
 22. 9. The isolatednucleic acid molecule of claim 1, comprising the nucleotide sequence ofSEQ ID NO:
 26. 10. Expression vector comprising the isolated nucleicacid molecule of claim 1, operably linked to a promoter.
 11. Eukaryoticcell line or prokaryotic cell strain, transformed or transfected withthe expression vector of claim
 10. 12. Isolated cancer associatedantigen comprising all or part of the amino acid sequence encoded by SEQID NO: 1, 3, 4, 8, 15, 19, 22 or
 26. 13. Eukaryotic cell line orprokaryotic cell strain, transformed or transfected with the isolatednucleic acid molecule of claim
 1. 14. The eukaryotic cell line orprokaryotic cell strain of claim 13, wherein said cell line is alsotransfected with a nucleic acid molecule coding for a cytokine.
 15. Theeukaryotic cell line or prokaryotic cell strain of claim 14, whereinsaid cell line is further transfected by a nucleic acid molecule codingfor an MHC molecule.
 16. The eukaryotic cell line or prokaryotic cellstrain of claim 14, wherein said cytokine is an interleukin.
 17. Theeukaryotic cell line or prokaryotic cell strain of claim 16, whereinsaid interleukin is IL-2, IL-4 or IL-12.
 18. The eukaryotic cell line orprokaryotic cell strain of claim 13, wherein said cell line has beenrendered non-proliferative.
 19. The eukaryotic cell line of claim 13,wherein said cell line is a fibroblast cell line.
 20. Expression vectorcomprising a mutated or attenuated virus and the isolated nucleic acidmolecule of claim
 1. 21. The expression vector of claim 20, wherein saidvirus is adenovirus or vaccinia virus.
 22. The expression vector ofclaim 21, wherein said virus is vaccinia virus.
 23. The expressionvector of claim 21, wherein said virus is adenovirus.
 24. Expressionsystem useful in transfecting a cell, comprising (i) a first vectorcontaining a nucleic acid molecule which codes for the isolated cancerassociated antigen of claim 13 and (ii) a second vector selected fromthe group consisting of (a) a vector containing a nucleic acid moleculewhich codes for an MHC or HLA molecule which presents an antigen derivedfrom said cancer associated antigen and (b) a vector containing anucleic acid molecule which codes for an interleukin.
 25. Immunogeniccomposition comprising the isolated cancer antigen of claim 12, and apharmaceutically acceptable adjuvant.
 26. The immunogenic composition ofclaim 25, wherein said adjuvant is a cytokine, a saponin, or GM-CSF. 27.Immunogenic composition comprising at least one peptide consisting of anamino acid sequence of from 8 to 12 amino acids concatenated to eachother in the isolated cancer associated cancer antigen of claim 12, anda pharmaceutically acceptable adjuvant.
 28. The immunogenic compositionof claim 27, wherein said adjuvant is a saponin, a cytokine, or GM-CSF.29. The immunogenic composition of claim 25, wherein said compositioncomprises a plurality of peptides which complex with a specific MHCmolecule.
 30. Immunogenic composition which comprises at least oneexpression vector which encodes a peptide derived from the amino acidsequence encoded by SEQ ID NO: 1, 3, 4, 8, 15, 19, 22 or
 26. 31. Theimmunogenic composition of claim 30, wherein said at least oneexpression vector codes for a plurality of peptides.
 32. Vaccine usefulin treating a subject afflicted with a cancerous condition comprisingthe isolated eukaryotic cell line of claim 13 and a pharmacologicallyacceptable adjuvant.
 33. The vaccine of claim 32, wherein saideukaryotic cell line has been rendered non-proliferative.
 34. Thevaccine of claim 33, wherein said eukaryotic cell line is a human cellline.
 35. A composition of matter useful in treating a cancerouscondition comprising a non-proliferative cell line having expressed onits surface a peptide derived from the amino acid sequence encoded bySEQ ID NO: 1, 3, 4, 8, 15, 19, 22 or
 26. 36. The composition of matterof claim 35, wherein said cell line is a human cell line.
 37. Acomposition of matter useful in treating a cancerous condition,comprising (i) a peptide derived from the amino acid sequence encoded bySEQ ID NO: 1, 3, 4, 8, 15, 19, 22 or 26, (ii) an MHC or HLA molecule,and (iii) a pharmaceutically acceptable carrier.
 38. Isolated antibodywhich is specific for the cancer associated antigen of claim
 12. 39. Theisolated antibody of claim 38, wherein said antibody is a monoclonalantibody.
 40. Method for screening for cancer in a sample, comprisingcontacting said sample with a nucleic acid molecule which hybridizes toall or part of the molecule encoded by SEQ ID NO: 1, 2, 3, 4, 8, 15, 19,22 or 26 and determining hybridization as an indication of cancer cellsin said sample.
 41. A method for screening for cancer in a sample,comprising contacting said sample with the isolated antibody of claim38, and determining binding of said antibody to a target as an indicatorof cancer.
 42. Method for diagnosing a cancerous condition in a subject,comprising contacting an immune reactive cell containing sample of saidsubject to a cell line transfected with the isolated nucleic acidmolecule of claim 1, and determining interaction of said transfectedcell line with said immunoreactive cell said interaction beingindicative of said cancer condition.
 43. A method for determiningregression, progression of onset of a cancerous condition comprisingmonitoring a sample from a patient with said cancerous condition for aparameter selected from the group consisting of (i) a protein encoded bySEQ ID NO: 1, 2, 3, 4, 8, 15, 19, 22 or 26, (ii) a peptide derived fromsaid protein, (iii) cytolytic T cells specific for said peptide and anMHC molecule with which it non-covalently complexes, and (iv) antibodiesspecific for said CT protein, wherein amount of said parameter isindicative of progression or regression or onset of said cancerouscondition.
 44. The method of claim 43, wherein said sample is a bodyfluid or exudate.
 45. The method of claim 43, wherein said sample is atissue.
 46. The method of claim 43, comprising contacting said samplewith an antibody which specifically binds with said protein or peptide.47. The method of claim 46, wherein said antibody is labelled with aradioactive label or an enzyme.
 48. The method of claim 46, wherein saidantibody is a monoclonal antibody.
 49. The method of claim 43,comprising amplifying RNA which codes for said protein.
 50. The methodof claim 49, wherein said amplifying comprises carrying out polymerasechain reaction.
 51. The method of claim 42, comprising contacting saidsample with a nucleic acid molecule which specifically hybridizes to anucleic acid molecule which codes for or expresses said protein.
 52. Themethod of claim 49, wherein said nucleic acid molecule comprises SEQ IDNO: 9, 10, 11, 12, 13, 14, 17, 18, 20, 21, 24, 25, 28 or
 29. 53. Themethod of claim 43, comprising assaying said sample for shed protein.54. The method of claim 43, comprising assaying said sample forantibodies specific for said protein, by contacting said sample withprotein.
 55. Method for diagnosing a cancerous condition comprisingassaying a sample taken from a subject for an immunoreactive cellspecific for a peptide derived from a protein encoded by SEQ ID NO: 1,2, 3, 4, 8, 15, 19, 22 or 26, complexed to an MHC molecule, presence ofsaid immunoreactive cell being indicative of said cancerous condition.56. Composition comprising at least one peptide consisting of an aminoacid sequence of from 8 to 25 amino acids concatenated to each other inthe isolated cancer associated antigen of claim 12, and apharmaceutically acceptable adjuvant.
 57. The composition of claim 56,wherein said adjuvant is a saponin, a cytokine, or GM-CSF.
 58. Thecomposition of claim 56, comprising a plurality of MHC binding peptides.59. Composition comprising an expression vector which encodes at leastone peptide consisting of an amino acid sequence of from 8 to 25 aminoacids concatenated to each other in the isolated cancer associatedantigen of claim 12, and pharmaceutically acceptable adjuvant.
 60. Thecomposition of claim 59, wherein said expression vector encodes aplurality of peptides.
 61. A method for screening for possible presenceof a pathological condition, comprising assaying a sample from a patientbelieved to have a pathological condition for antibodies specific to atleast one of the cancer associated antigens encoded by SEQ ID NOS: 1, 2,3, 4, 8, 15, 19, 22 or 26, presence of said antibodies being indicativeof possible presence of said pathological condition.
 62. The method ofclaim 61, wherein said pathological condition is cancer.
 63. The methodof claim 61, wherein said cancer is melanoma.
 64. The method of claim61, further comprising contacting said sample to purified cancerassociated antigen encoded by SEQ ID NO: 1, 3, 4, 8, 15, 19, 22 or 26.65. A method for screening for possible presence of a pathologicalcondition in a subject, comprising assaying a sample taken from saidsubject for expression of a nucleic acid molecule, the nucleotidesequence of which comprises SEQ ID NO: 1, 2, 3, 4, 8, 15, 19, 22 or 26,expression of said nucleic acid molecule being indicative of possiblepresence of said pathological condition.
 66. The method of claim 65,wherein said pathological condition is cancer.
 67. The method of claim65, comprising determining expression via polymerase chain reaction. 68.The method of claim 65, comprising determining expression by contactingsaid sample with at least one of SEQ ID NO: 9, 10, 11, 12, 13, 14, 17,18, 20, 21, 24, 25, 28 or
 29. 69. A method for determining regression,progression of onset of a cancerous condition comprising monitoring asample from a patient with said cancerous condition for a parameterselected from the group consisting of (i) a cancer associated antigenencoded by SEQ ID NO: 1, 2, 3, 4, 8, 15, 19, 22 or 25, (ii) a peptidederived from said cancer associated antigen, (iii) cytolytic T cellsspecific for said peptide and an MHC molecule with which itnon-covalently complexes, and (iv) antibodies specific for said cancerassociated antigen, wherein amount of said parameter is indicative ofprogression or regression or onset of said cancerous condition.
 70. Themethod of claim 69, wherein said sample is a body fluid or exudate. 71.The method of claim 69, wherein said sample is a tissue.
 72. The methodof claim 69, comprising contacting said sample with an antibody whichspecifically binds with said protein or peptide.
 73. The method of claim72, wherein said antibody is labelled with a radioactive label or anenzyme.
 74. The method of claim 72, wherein said antibody is amonoclonal antibody.
 75. The method of claim 69, comprising amplifyingRNA which codes for said protein.
 76. The method of claim 75, whereinsaid amplifying comprises carrying out polymerase chain reaction. 77.The method of claim 69, comprising contacting said sample with a nucleicacid molecule which specifically hybridizes to a nucleic acid moleculewhich codes for or expresses said protein.
 78. The method of claim 69,comprising assaying said sample for shed cancer associated antigen. 79.The method of claim 69, comprising assaying said sample for antibodiesspecific for said cancer associated antigen, by contacting said samplewith said cancer associated antigen.
 80. Method for screening for acancerous condition comprising assaying a sample taken from a subjectfor an immunoreactive cell specific for a peptide derived from a cancerassociated antigen encoded by SEQ ID NO: 1, 2, 3, 4, 8, 15, 19, 22 or26, complexed to an MHC molecule, presence of said immunoreactive cellbeing indicative of said cancerous condition.
 81. An isolated nucleicacid molecule consisting of a nucleotide sequence defined by SEQ ID NO:1, 2, 3, 8, 15, 19, 22 or
 26. 82. Isolated nucleic acid molecule thecomplimentary sequence of which hydridizes, under stringent conditions,to the nucleotide sequence set forth in SEQ ID NO: 4, 5, 8, 15, 19, 22or
 26. 83. An isolated polypeptide comprising at least 9 consecutiveamino acids set forth in SEQ ID NO: 5, 7, 16, 19, 23, 27, or
 30. 84. Theisolated polypeptide of claim 83, comprising at least 9 consecutiveamino acids set forth in SEQ ID NO: 23 or
 30. 85. The isolatedpolypeptide of claim 84, comprising t least 9 consecutive amino acids ofthe amino acid sequence set forth in SEQ ID NO:
 23. 86. The isolatedpolypeptide of claim 85, comprising amino acids 102-111, 904-912 or1262-1270 of SEQ ID NO:
 23. 87. An isolated nucleic acid molecule whichencodes the amino acid sequence of SEQ ID NO:
 30. 88. An isolatednucleic acid molecule which encodes the isolated polypeptide of claim86.
 89. Expression vector comprising the isolated nucleic acid moleculeof claim 88, operably linked to a promater.